SESSION 1 JOINT OPENING SESSION

WELCOME

SHAW P

Chair of the Symposium Programme Committee, Academic Neurology Unit, University of Sheffield, United Kingdom

COMMUNICATING SCIENCE TO POLITICIANS AND THE PUBLIC

Blakemore C

Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom

SESSION 2A CELL BIOLOGY & PATHOLOGY

C1 THE BLOOD-BRAIN BARRIER AND ITS RELEVANCE TO PATHOGENESIS AND THERAPY IN NEURODEGENERATIVE DISEASE

ABBOTT J

King's College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: blood brain barrier; blood-spinal cord barrier

The blood-brain barrier (BBB) is formed by the endothelial cells lining the cerebral microvessels; the blood-spinal cord barrier (BSCB) is the equivalent structure in the spinal cord. The chief characteristic of these barriers is the extreme tightness of the zipper-like ‘tight junctions’ that link neighbouring endothelial cells, making the vessels in the central nervous system (CNS) around 50–100 times tighter than those of peripheral tissues such as muscle and skin.

The tight barrier significantly reduces the ability of water soluble compounds to diffuse into or out of the brain via the junctional cleft (paracellular pathway), so that most molecular traffic across the endothelium is via the cells (transcellular pathway). Small gaseous molecules such as oxygen and carbon dioxide can diffuse freely through the cell membranes, as can lipid soluble agents such as ethanol and many CNS drugs. Water-soluble molecules the brain needs such as glucose and amino acids can be carried across the endothelium on specific ‘transporters’, and efflux transporters help exclude or get rid of toxic agents and metabolites, but also exclude some potentially useful CNS drugs.

The barriers are critical to normal healthy function of the CNS, especially by maintaining a constant central environment for neuronal activity. The perivascular space around the microvessels bounded by the endfeet of astrocytic glia has a further protective role, by providing a ‘niche’ for elements of the cellular immune system.

Disturbances in CNS barrier function are present in a number of neurodegenerative disorders (NDDs), including multiple sclerosis, Alzheimer's disease and stroke; they can contribute to exacerbation of symptoms and neural damage, and may also play a causative role under some conditions. There is growing recognition that CNS inflammation is an element in many of these disorders, with the BBB, BSCB and perivascular niche being critical sites for the inflammatory process.

The evidence for BBB/BSCB involvement in ALS is relatively new, from observations in patients, and from animal models especially the G93A mutation of superoxide dismutase SOD1. Changes reported include leakage of plasma proteins into the CNS and cerebrospinal fluid (CSF), compromised endothelial tight junctions, and changes in vascular anatomy and blood flow. Thus for ALS, as for many other NDDs, the endothelium of CNS microvessels may be a promising target for drug therapies, to maintain endothelial and barrier health, and to repair the dysfunctional barrier.

More research is needed, sampling CSF and plasma to follow the course of barrier disturbance in human disease, animal experimentation to reveal features of the barriers in vivo, and cell culture models to allow detailed studies of the mechanisms of action at the cellular and molecular level. The CNS endothelium may prove a useful future target for ALS therapy.

C2 A NEW ROLE FOR ANGIOGENIN IN NEURITE PATHFINDING AND SURVIVAL- IMPLICATIONS FOR ALS

SUBRAMANIAN V, CRABTREE B, FENG Y, ACHARYA R

Department of Biology and Biochemistry, University of Bath, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Angiogenin, pluripotent stem cells, neuroprotection

Introduction: Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder affecting upper and lower motor neurons (MNs) with fatal consequences. The molecular mechanisms underlying ALS are poorly understood and mutations in SOD1 is one of the known causes of ALS. However, mutations in SOD1 are seen only in a very small number of cases of ALS. Interestingly, mutations in human angiogenin (hANG), a member of the ribonuclease A (RNase A) superfamily known to be involved in neovascularization, have been recently reported in patients with ALS [1–4] but the expression and function of Ang in the nervous system and the effects of these mutations on MN differentiation and survival has not been investigated.

Objectives: The main objectives are 1) to study the expression of Ang during mouse embryogenesis 2) to investigate its importance in neuronal differentiation 3) to study the effects of the hAng-ALS variants on neurons.

Methods: The distribution of Ang in the mouse developing nervous system was investigated by immuno-histochemistry. The pluripotent embryonal carcinoma cell culture model of neuroectodermal differentiation was used to study the expression of Ang during MN differentiation by immunocytochemistry and effect of a small molecule inhibitor of Ang on neuronal differentiation was investigated. The hANG-ALS variants were generated by site directed mutagenesis and the proteins were expressed and purified. The effects of hANG and hAng-ALS variants on MN differentiation, neurite extension as well as their ability to protect MNs from hypoxia induced cell death was investigated using the cell culture model.

Results: Mouse angiogenin-1 (mAng-1) is strongly expressed in the developing nervous system in mouse embryogenesis and during neural differentiation of pluripotent P19 embryonal carcinoma cells. NCI 65828, a cell permeable inhibitor of hANG inhibits neurite extension/pathfinding by P19 derived neurons without affecting their differentiation to the neuronal lineage [5]. We also report that neurite extension/pathfinding by pluripotent EC cell derived neurons treated with the hANG ALS mutants is compromised. hANG ALS mutants also have a cytotoxic effect on MNs leading to their degeneration. hANG was able to protect neurons from hypoxia induced cell death but the mutants of hANG lacked neuroprotective activity.

Discussion and Conclusions: Our findings show that ANG plays an important role in neurite pathfinding and survival providing the first causal link between mutations in hANG and ALS.

C3 RETINOID SIGNALING ALTERATIONS IN ALS AND THE CONSEQUENCES OF THESE ALTERATIONS IN MOTOR NEURON-ENRICHED CULTURES

KOLARCIK C, BOWSER R

University of Pittsburgh School of Medicine, Pennsylvania, United States

E-mail address for correspondence: [email protected]

Keywords: retinoid signaling, RARbeta, motor neurons

Background: Previous reports demonstrated decreased transthyretin (TTR) levels in the cerebrospinal fluid of ALS patients. TTR plays multiple physiological roles and functions within the central nervous system to regulate protein aggregation, antioxidant activity and the retinoid signaling pathway. Multiple studies have described the differential expression of genes regulated by retinoid signaling in post-mortem tissues from individuals with ALS. Similarly, alterations in retinoid-regulated gene expression have been observed in transgenic animal models of ALS prior to disease-related morphological changes and symptom onset.

Objectives: Our overall objectives were to identify and then explore the functional consequences of alterations of the retinoid signaling pathway in ALS to test the hypothesis that retinoid signaling is deficient in ALS and that it contributes to the motor neuron cell death that occurs in the disease.

Methods: Using post-mortem lumbar spinal cord tissue, we characterized the expression and distribution of retinoid signaling components with immunohistochemistry, immunoblotting and confocal microscopy. An in vitro motor neuron-enriched cell culture system was then used to perform more functional studies in which we focused on the nuclear genomic and non-genomic effects of retinoic acid signaling in motor neurons.

Results: Differences in cytoplasmic binding proteins including cellular retinol binding protein (CRBP) and cellular retinoic acid binding proteins (CRABP)-I and II were observed. CRBP immunoreactivity was higher in ALS spinal cord motor neurons while CRABP-I was decreased. CRABP–II was concentrated to the nucleus in ALS motor neurons while control motor neurons exhibited a diffuse cytoplasmic pattern. We also assessed the expression and distribution of retinoic acid nuclear receptors. RARβ exhibited increased immunoreactivity in motor neuron nuclei of individuals with sporadic ALS although both controls and individuals with familial ALS lacked this punctate nuclear immunostaining. In our in vitro system, we modulated individual nuclear receptors with agonists and antagonists to further characterize their effects on gene transcription and simultaneously evaluated the more rapid, non-genomic effects by assessing phosphatidylinositol-3-kinase (PI3K) and ERK1/2 MAPK signaling pathways.

Conclusions: Our results indicate that retinoic acid signaling is altered in ALS and components of this pathway may represent novel therapeutic targets. In particular, the differences we have observed with respect to RARβ suggest that retinoid signaling impacts only the sporadic form of the disease while it does not play a role in familial cases. This may indicate differences between the two forms of the disease.

C4 METABOLIC DIFFERENCES BETWEEN BRAIN AND SPINAL CORD MITOCHONDRIA OF WILD TYPE AND HUMAN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS MUTANT SOD1-TRANSGENIC RATS

PANOV A, KUBALIK N, HEMENDINGER R, BROOKS B

Carolinas Medical Center, Charlotte, North Carolina, United States

E-mail address for correspondence: [email protected]

Keywords: mitochondria, calcium, ROS, metabolism

Background.ALS is associated with hypermetabolism and mitochondrial dysfunction [1], [2]. In SOD1 during the early pre-symptomatic stage, many spinal cord mitochondria (SCM) in large myelinated axons exhibit swelling with an increased number of cristae, and show small vacuoles in the matrix, cristae or both [3]. With neuronal activation, brain mitochondria (BM) and SCM utilize a mixture of substrates: glutamate, pyruvate and malate, with increased ATP production. However, in resting BM and SCM, oxidation of glutamate, pyruvate and malate increased ROS generation due to increased reverse electron transport (RET).

Objectives: To determine, quantitatively with our novel methodology, differences between BM and SCM from wild type (WT) and SOD1 rats in major mitochondrial functions: oxidative phosphorylation, permeability transition and mechanisms of ROS generation.

Methods: Non-synaptic BM and SCM from 8 week old WT and SOD1 rats were analyzed for tissue Ca^2 + content by atomic absorption, respiration by Mitocell S200 Micro Respirometry System and ROS by Amplex Red.

Results: In comparison with WT, in SOD1 the yields of BM were diminished by 20% and SCM by 57%. The respiratory activities were also significantly diminished with all substrate mixtures, particularly in SOD1 SCM. The Ca^2 + content in the WT spinal cord was 8 fold larger than in WT brain. The calcium retention capacity (CRC) of BM from WT exceeded 4-fold the total Ca^2 + content in WT brain (both per 1g of wet tissue). The CRC of WT SCM corresponded to only 10% of the total WT spinal cord Ca^2 + content. Both WT and SOD1 SCM showed much higher rates of succinate oxidation than WT and SOD1 BM. Despite significantly diminished rates of state 4 respiration, SCM and BM from SOD1 rats showed several-fold higher rates of ROS generation with glutamate, pyruvate and malate which was sensitive to malonate.

Conclusions: SCM have significantly lower intrinsic inhibition of succinate oxidation, which resulted in higher rates of the RET-driven ROS generation as compared with BM from both WT and particularly SOD1 rats. Loss of mitochondria in spinal cord of SOD1 rats begins long before neurological dysfunction. These data suggest that poorly buffered Ca^2 + released during the pre-symptomatic phase of neuroaxonal degeneration, together with increased ROS generation may be responsible for early loss of SCM in SOD1 rats.

C5 DIFFERENTIALLY EXPRESSED BIOLOGICAL PROCESSES IN RELEVANT SPINAL COMPARTMENTS ISOLATED BY MICRODISSECTION IN SOD1 TRANSGENIC MICE

RAVITS J¹, FAN Y¹, RABIN S¹, STONE B¹, BEYER D², BAMMLER T², BUMGARNER R², LASPADA AL²

¹Benaroya Research Institute, Seattle, ²University of Washington, Seattle, WA, United States

E-mail address for correspondence: [email protected]

Keywords: Genomics, gene expression, microarray

Background: There are now three published whole genome expression profiles of laser captured motor neurons. We performed similar studies except that we also: (a) profiled anterior horns as well as motor neurons; (b) used 2 different controls; (c) looked at very early time points; and (d) analyzed biological processes as well as single genes.

Objectives: To profile early differentially expressed biological processes in the cellular compartments relevant to motor neuron degeneration in G93A mouse model of ALS.

Methods: We used G93A mice and 2 controls (G93A littermates and human wild type transgenic mice). We studied the mice at 20 and 60 days. We created 2 separate RNA pools, 1 enriched with motor neurons isolated by laser capture microdissection and 1 enriched with anterior horns collected after removal of motor neurons. We synthesized a cDNA probe using in vitro transcription amplification. We profiled gene expression using whole genome oligonucleotide microarray. We processed microarray data for biological enrichment using Gene Set Analysis (GSA) (www-stat.stanford.edu/∼tibs/GSA/), which profiles differentially expressed sets of genes representing biological processes defined in Gene Ontology rather than just differentially expressed individual genes.

Results: In the motor neuron compartment at 20 days: there was up-regulation of purine nucleoside monophosphate biology, negative regulation of B-cell activation, and thyroid hormone metabolism; there was down-regulation of adenylate cyclase activation, amino acid derivative metabolism, peptide hormone processing, and positive regulation of lyase activity. At 60 days: there was up-regulation of serine metabolism and DNA damage response, signal transduction resulting in induction of apoptosis; there was down-regulation of apoptotic nuclear changes, aspartate family amino acid metabolism, cholesterol/sterol biosynthesis, regulation of B cell proliferation, DNA catabolism, and G-M transition of mitotic cell cycle. In the anterior horn compartment at 20 days: there was up-regulation of glutamate metabolism, JAK-STAT cascade, regulation of amino acid metabolism, regulation of phosphate metabolism and regulation of phosphorylation, regulation of protein amino acid phosphorylation, and positive regulation of peptidyl-tyrosine phosphorylation; there was down-regulation of adenylate cyclase biology, regulation of lyase activity, phospholipid catabolism, peptide hormone processing, and mechanosensory behavior. At 60 days: there was up-regulation of carbohydrate biosynthesis and gluconeogenesis; there was down-regulation of negative regulation of Wnt receptor signalling pathway, neurogenesis, neuron differentiation, positive regulation of B cell proliferation, and positive regulation of JNK cascade.

Discussion/Conclusions: GSA allows the discovery-based power of microarray to be carried into data interpretation. The deregulated biological processes were distinctly resolved but very different between the motor neuron and anterior horn compartments, as stated above. The processes deregulated at 20 days did not remain deregulated at 60 days. There were marked differences just comparing the 2 different controls to each other and marked improvement in the signal resolution in disease by using both. Data interpretation still remains the biggest challenge for these studies.

C6 GENE EXPRESSION PROFILING TO INVESTIGATE THE STRESS EFFECTS OF PHYSICAL EXERCISE ON THE MOTOR NEURONE TRANSCRIPTOME

FERRAIUOLO L¹, DE BONO J², HEATH P¹, HOLDEN H¹, KASHER P¹, CHANNON K², KIRBY J¹, SHAW P¹

¹University of Sheffield, United Kingdom, ²University of Oxford, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: exercise, motor neurons, microarrays

Background: A body of evidence indicates that physical activity is implicated as a risk factor in ALS: 1. Well known sportsman such as Lou Gehrig (baseball) and Donald Levey (football) have developed MND; 2. In several studies, participation in sports/athleticism has been identified as a risk factor; 3. Recent reports have shown an increased incidence of ALS in Italian and American soccer players. The hypothesis has been put forward that a high level of physical exercise is associated with an increased risk of developing ALS in individuals with an underlying genetic susceptibility, which may result in a failure to mount the normal physiological response to physical exercise. We have investigated the transcriptome of motor neurones (MN), the vulnerable cell population in MND, and muscles in response to exercise.

Objective: Our aim is to identify 1) which genes are activated/repressed in response to exercise in muscles and MN in absence of other stress; 2) which genes belong to a specific motor neuronal response; 3) which cellular compartments are under stress during physical training and whether these can be involved in the pathophysiology of ALS.

Methods: Gastrocnemius muscle and approximately 1000 MN have been isolated from lumbar spinal cord of 3 female mice undergoing voluntary exercise (mean running distance of 13km per day) for 21±1 days and 3 sedentary mice. Muscular RNA was isolated using Quiazol kit (Quiagen); motor neuronal RNA was extracted using Picopure kit (Arcturus), amplified using the Affymetrix Amplification kit (Arcturus) and labelled using the GeneChip Expression IVT Labelling Kit (Affymetrix). 10 µ g cRNA was applied to the Affy Mouse Genome 430 2.0 GeneChip, and data analysis was performed using ArrayAssist (Iobion).

Results: After 3 weeks of voluntary exercise, analysis of the motorneuronal transcriptome showed upregulation of 203 transcripts and downregulation of 241. The main changes affect genes encoding for neurotrophic factors and their receptors, i.e. ciliary neurotrophic factor, leukaemia inhibitor factor receptor and activin receptor 2a; genes involved in neurotransmitter release and regulation of ion currents and membrane potential, i.e. K⁺ and Ca^2 + channels, along with genes involved in the regulation of NMDA receptor expression at the synapse, i.e. the splicing factor Nova2. Mechanisms such as branching process and transcription modulation are also altered. In gastrocnemius muscle 194 genes were upregulated and 176 downregulated. The main changes affect angiogenesis, favoured by upregulation of VEGF receptor 2 and Epas1; myogenesis, stimulated by increased levels of Trkb and Hdgf and ECM reorganisation.

Conclusions: Our study highlights some important similarities between the physiological response of MN and skeletal muscle to exercise and the pathophysiology of ALS. 1. The key role of Cntf, Lif and Vegfr2. 2. The potential deregulation of K⁺ channels as cause of the abnormal membrane electric properties affecting ALS patients and their balancing function in exercise. 3. Axonal growth and branching process, likely to be altered in ALS, are fundamental in the reinforcement of the NMJ in response to exercise. Functional polymorphism studies on candidate genes in ALS patients will be used to investigate the relation between the mechanisms altered in response to exercise and the pathophysiology of ALS.

C7 AN AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTATION IN VAPB IMPAIRS AXONAL TRANSPORT OF MITOCHONDRIA

DE VOS KJ¹, TUDOR EL¹, LAU KF², ACKERLEY S¹, LEIGH PN¹, SHAW CE¹, MILNER CC J¹

¹MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College, London, United Kingdom, ²Department of Biochemistry, The Chinese University of Hong Kong, China

E-mail address for correspondence: [email protected]

Keywords: Axonal transport, Mitochondria, VAPB

Background: A mutation in vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes familial ALS but the mechanisms whereby VAPBP56S induces disease are not fully understood. We have previously shown that disruption to axonal transport is a very early event in mutant superoxide dismutase 1 (SOD1) induced ALS [1]. The early disruption of axonal transport suggests that compromised axonal transport of selected cargoes may be causative rather than secondary in ALS.

Objectives: The objective of this study was to analyze the effect that VAPBP56S has on axonal transport of mitochondria.

Methods: Mouse cortical neurons were transfected with empty vector, wild-type VAPB or VAPBP56S and axonal transport of mitochondria labeled with mitochondria-targeted red fluorescent protein was quantified from time-lapse recordings as described previously [1].

Results: VAPBP56S significantly decreased the total number of motile mitochondria by specific reduction of the number of anterogradely-transported mitochondria; net retrograde transport of mitochondria was unaffected. This specific inhibition of net anterograde transport significantly shifted the balance of axonal transport of mitochondria. Detailed quantification of the mitochondrial transport activity showed that VAPBP56S induced a significant decrease in anterograde transport activity but did not affect retrograde transport activity.

The velocity of transport is an intrinsic property of the molecular motors that drive transport. Hence to assess any direct damage to molecular motors, the transport velocity was determined by tracking individual mitochondria through axons. VAPBP56S did not affect anterograde or retrograde transport velocity indicating that molecular motors were not damaged.

Conclusion: These observations suggest that VAPBP56S specifically inhibits anterograde transport of mitochondria by affecting the regulation of the anterograde motor kinesin-1. Interestingly, the nature of this impairment of anterograde transport and the resulting imbalance of mitochondrial transport is very similar to that caused by ALS mutant SOD1 [1].

Our findings provide new insights into the mechanisms by which VAPBP56S induces motor neuron demise. Furthermore, our analyses reveal that disturbance of anterograde mitochondrial transport is a common feature in at least 2 distinct forms of familial ALS. As such, correcting axonal transport defects represent a promising therapeutic target.

C8 SPASTIN MUTATIONS DISRUPT AXONAL TRANSPORT IN HEREDITARY SPASTIC PARAPLEGIA (HSP)

KASHER P¹, DE VOS K², BINGLEY M¹, MILNER R¹, MCDERMOTT C¹, WHARTON S¹, WOOD J¹, SHAW P¹, GRIERSON A¹

¹University of Sheffield, United Kingdom, ²Kings College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: spastin, axonal, transport

Background: Mutations in the spastin gene, on the SPG4 locus, are the most prevalent cause of HSP, accounting for approximately 40% of cases. Spastin is a microtubule severing protein, and several groups have demonstrated that both its knockdown and the overexpression of disease-causing mutants result in axonal defects. We hypothesise that spastin mutations directly disrupt axonal transport.

Objectives: To gain insight into how mutations in spastin lead to axonal degeneration in HSP by quantification of axonal transport of mitochondria in neurons in the presence or absence of mutant spastin.

Methods: Mice harbouring a point mutation in a splice donor site in the SPG4 gene were identified and re-derived onto a C57BL/6 background. Primary cortical neurons were prepared from E15.5 homozygous and heterozygous mutant spastin mice and wild-type littermate controls. Immunocytochemistry was performed on fixed cultures to examine the presence of axonal swellings, using antibodies raised against cytoskeletal and vesicular cargoes. Mouse cortical neurons were co-transfected with DsRed2Mito and EGFP-empty vector. Live cellular imaging was performed and various properties of mitochondrial motility were analysed. To determine the presence of axonal swellings in adult mice, immunohistochemistry was performed on cervical and lumbar spinal cord sections from homozygous and heterozygous mutant spastin mice of various ages and wild-type littermate controls to identify axonal swellings. Similar axonal transport data were recorded in rat primary cortical neurons co-transfected with DsRed2Mito and either EGFP-spastin wild type, EGFP-spastin K388R, or EGFP empty vector.

Results: We show that the mouse spastin mutation effects mRNA splicing and causes the creation of a premature stop codon. Axonal swellings occur in cultured mutant spastin neurons. Swellings are associated with significant accumulations of a number of cytoskeletal and vesicular cargoes. These cargo accumulations were also identified in the corticospinal tract of adult mice. Specific properties of both anterograde and retrograde transport were disrupted in the presence of mutant spastin. We demonstrate that swellings are dynamic structures as mitochondria, although often stalled temporarily, were able to enter and leave these structures. In transfected rat neurons the K388R spastin mutant caused comparable defects in axonal transport.

Discussion: Together these results demonstrate that 2 different types of spastin mutation directly disrupt axonal transport, and are likely to play an important role in the dying back axonopathy, which is the principal pathological feature of HSP. As the axonal transport defects and the abnormal accumulation of cytoskeleton and vesicular cargoes are evident in the heterozygous mutant spastin mice, we promote the current spastin mouse model as an effective tool to study SPG4-linked HSP pathophysiology.